The broad aim of this project is to understand how the cardiac myocyte differentiation program is activated during vertebrate development. In previous work the principal investigator has identified a cardiac-inducing activity in the anterior lateral plate endoderm of the gastrula-stage avian embryo, which can induce cells from the posterior primitive streak (which normally gives rise to extra embryonic tissues and to blood) to give rise to cardiac myocytes. Recently, the principal investigator has extended these studies to demonstrate that a combination of at least two signals is necessary to induce cardiac myocyte determination. During mid-primitive streak stage in chick embryos, cells which contact anterior endoderm seem to lie in a developmental field in which cells display a potential to become heart. BMP-2 and/or BMP-4 then appear to be capable of inducing cells within this field to initiate the cardiac differentiation program. One of the first cardiac markers that the principal investigator has found to be induced in primitive streak cells exposed to heart inducing signals is the homeobox-containing gene, Nkx-2.5. In Drosophila, the homologous gene tinman is expressed in the heart and is required for heart formation. Work from the principal investigator's lab suggests that a combination of at least two signals, a cardiac promoting signal from the anterior endoderm plus a BMP-2/-4 signal, are together necessary to initiate expression of the Nkx-2.5 gene. The goal of this proposal is to identify the sequence elements in the Nkx-2.5 gene that are responsive to these signaling molecules and the transcription factors that induce cardiac-specific expression of this gene. Aim 1. Map the region of the chick Nkx-2.5 gene that responds to cardiac inducing signals in an in vitro cardiac induction assay. Aim 2. Identify the transcription factors that bind to the cardiac responsive sequences in the chick Nkx-2.5 gene. Aim 3. Determine if a chick Mad1 family member expressed in the precardiac mesoderm can mediate the cardiac inducing properties of BMP-2/-4. Aim 4. Identify transcription factors that mediate activation of Nkx-2.5 in response to BMP signals by screening for proteins that interact with chick Mad1 in precardiac mesoderm.